Activation of Both MAP Kinase and Phosphatidylinositide 3-Kinase by Ras Is Required for Hepatocyte Growth Factor/Scatter Factor–induced Adherens Junction Disassembly

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of colonies followed by disruption of cell–cell junctions and subsequent cell scattering. In Madin–Darby canine kidney cells, HGF/SF-induced motility involves actin reorganization mediated by Ras, but whether Ras and downstream signals regulate the breakdown of intercellular adhesions has not been established. Both HGF/SF and V12Ras induced the loss of the adherens junction proteins E-cadherin and β-catenin from intercellular junctions during cell spreading, and the HGF/SF response was blocked by dominant-negative N17Ras. Desmosomes and tight junctions were regulated separately from adherens junctions, because they were not disrupted by V12Ras. MAP kinase, phosphatidylinositide 3-kinase (PI 3-kinase), and Rac were required downstream of Ras, because loss of adherens junctions was blocked by the inhibitors PD098059 and LY294002 or by dominant-inhibitory mutants of MAP kinase kinase 1 or Rac1. All of these inhibitors also prevented HGF/SF-induced cell scattering. Interestingly, activated Raf or the activated p110α subunit of PI 3-kinase alone did not induce disruption of adherens junctions. These results indicate that activation of both MAP kinase and PI 3-kinase by Ras is required for adherens junction disassembly and that this is essential for the motile response to HGF/SF.

Replication Factor C3 of Schizosaccharomyces pombe, a Small Subunit of Replication Factor C Complex, Plays a Role in Both Replication and Damage Checkpoints

We report here the isolation and functional analysis of the rfc3+ gene of Schizosaccharomyces pombe, which encodes the third subunit of replication factor C (RFC3). Because the rfc3+ gene was essential for growth, we isolated temperature-sensitive mutants. One of the mutants, rfc3-1, showed aberrant mitosis with fragmented or unevenly separated chromosomes at the restrictive temperature. In this mutant protein, arginine 216 was replaced by tryptophan. Pulsed-field gel electrophoresis suggested that rfc3-1 cells had defects in DNA replication. rfc3-1 cells were sensitive to hydroxyurea, methanesulfonate (MMS), and gamma and UV irradiation even at the permissive temperature, and the viabilities after these treatments were decreased. Using cells synchronized in early G2 by centrifugal elutriation, we found that the replication checkpoint triggered by hydroxyurea and the DNA damage checkpoint caused by MMS and gamma irradiation were impaired in rfc3-1 cells. Association of Rfc3 and Rad17 in vivo and a significant reduction of the phosphorylated form of Chk1 in rfc3-1 cells after treatments with MMS and gamma or UV irradiation suggested that the checkpoint signal emitted by Rfc3 is linked to the downstream checkpoint machinery via Rad17 and Chk1. From these results, we conclude that rfc3+ is required not only for DNA replication but also for replication and damage checkpoint controls, probably functioning as a checkpoint sensor.

T Cell Receptor-induced Activation and Apoptosis In Cycling Human T Cells Occur throughout the Cell Cycle

Previous studies have found conflicting associations between susceptibility to activation-induced cell death and the cell cycle in T cells. However, most of the studies used potentially toxic pharmacological agents for cell cycle synchronization. A panel of human melanoma tumor-reactive T cell lines, a CD8+ HER-2/neu-reactive T cell clone, and the leukemic T cell line Jurkat were separated by centrifugal elutriation. Fractions enriched for the G0–G1, S, and G2–M phases of the cell cycle were assayed for T cell receptor-mediated activation as measured by intracellular Ca2+ flux, cytolytic recognition of tumor targets, and induction of Fas ligand mRNA. Susceptibility to apoptosis induced by recombinant Fas ligand and activation-induced cell death were also studied. None of the parameters studied was specific to a certain phase of the cell cycle, leading us to conclude that in nontransformed human T cells, both activation and apoptosis through T cell receptor activation can occur in all phases of the cell cycle.

How well can an amoeba climb?

We report here our efforts to measure the crawling force generated by cells undergoing amoeboid locomotion. In a centrifuge microscope, acceleration was increased until amoebae of Dictyostelium discoideum were “stalled” or no longer able to “climb up.” The “apparent weight” of the amoebae at stalling rpm in myosin mutants depended on the presence of myosin II (but not myosins IA and IB) and paralleled the cortical strength of the cells. Surprisingly, however, the cell stalled not only in low-density media as expected but also in media with densities greater than the cell density where the buoyant force should push the amoeba upward. We find that the leading pseudopod is bent under centrifugal force in all stalled amoebae, suggesting that this pseudopod is very dense indeed. This finding also suggests that directional cell locomotion against resistive forces requires a turgid forward-pointing pseudopod, most likely sustained by cortical actomyosin II.

Plasticity of the cochleotopic (frequency) map in specialized and nonspecialized auditory cortices

Auditory conditioning (associative learning) causes reorganization of the cochleotopic (frequency) maps of the primary auditory cortex (AI) and the inferior colliculus. Focal electric stimulation of the AI also evokes basically the same cortical and collicular reorganization as that caused by conditioning. Therefore, part of the neural mechanism for the plasticity of the central auditory system caused by conditioning can be explored by focal electric stimulation of the AI. The reorganization is due to shifts in best frequencies (BFs) together with shifts in frequency-tuning curves of single neurons. In the AI of the Mongolian gerbil (Meriones unguiculatus) and the posterior division of the AI of the mustached bat (Pteronotus parnellii), focal electric stimulation evokes BF shifts of cortical auditory neurons located within a 0.7-mm distance along the frequency axis. The amount and direction of BF shift differ depending on the relationship in BF between stimulated and recorded neurons, and between the gerbil and mustached bat. Comparison in BF shift between different mammalian species and between different cortical areas of a single species indicates that BF shift toward the BF of electrically stimulated cortical neurons (centripetal BF shift) is common in the AI, whereas BF shift away from the BF of electrically stimulated cortical neurons (centrifugal BF shift) is special. Therefore, we propose a hypothesis that reorganization, and accordingly organization, of cortical auditory areas caused by associative learning can be quite different between specialized and nonspecialized (ordinary) areas of the auditory cortex.

Magnesium-Chelatase from Developing Pea Leaves1 : Characterization of a Soluble Extract from Chloroplasts and Resolution into Three Required Protein Fractions

Mg-chelatase catalyzes the ATP-dependent insertion of Mg2+ into protoporphyrin-IX to form Mg-protoporphyrin-IX. This is the first step unique to chlorophyll synthesis, and it lies at the branch point for porphyrin utilization; the other branch leads to heme. Using the stromal fraction of pea (Pisum sativum L. cv Spring) chloroplasts, we have prepared Mg-chelatase in a highly active (1000 pmol 30 min−1 mg−1) and stable form. The reaction had a lag in the time course, which was overcome by preincubation with ATP. The concentration curves for ATP and Mg2+ were sigmoidal, with apparent Km values for Mg2+ and ATP of 14.3 and 0.35 mm, respectively. The Km for deuteroporphyrin was 8 nm. This Km is 300 times lower than the published porphyrin Km for ferrochelatase. The soluble extract was separated into three fractions by chromatography on blue agarose, followed by size-selective centrifugal ultrafiltration of the column flow-through. All three fractions were required for activity, clearly demonstrating that the plant Mg-chelatase requires at least three protein components. Additionally, only two of the components were required for activation; both were contained in the flow-through from the blue-agarose column.

Evidence for a novel DNA damage binding protein in human cells.

We describe a novel DNA damage binding activity in nuclear extracts from a normal human fibroblast cell strain. This protein was identified using electrophoretic mobility shift assays of immunopurified UV-irradiated oligonucleotide substrates containing a single, site-specific cyclobutane pyrimidine dimer or a pyrimidine (6-4) pyrimidinone photoproduct. Compared with the (6-4) photoproduct, which displayed similar levels of binding in double and single-stranded substrates, the protein showed somewhat lower affinity for the cyclobutane dimer in a single-stranded oligonucleotide and negligible binding in double-stranded DNA. The specificity and magnitude of binding was similar in cells with normal excision repair (GM637) and repair-deficient cells from xeroderma pigmentosum groups A (XP12RO) and E (XP2RO). An apparent molecular mass of 66 kDa consisting of two subunits of approximately 22 and approximately 44 kDa was determined by Southwestern analysis. Cell cycle studies using centrifugal cell elutriation indicated that the binding activity was significantly greater in G1 phase compared with S phase in a human lymphoblast cell line. Gel supershift analysis using an anti-replication protein A antibody showed that the binding protein was not antigenically related to the human single-stranded binding protein. Taken together, these data suggest that this activity represents a novel DNA damage binding protein that, in addition to a putative role in excision repair, may also function in cell cycle or gene regulation.

Pluripotent hematopoietic stem cells contain high levels of mRNA for c-kit, GATA-2, p45 NF-E2, and c-myb and low levels or no mRNA for c-fms and the receptors for granulocyte colony-stimulating factor and interleukins 5 and 7.

Pluripotent hematopoietic stem cells (PHSCs) were highly enriched from mouse bone marrow by counterflow centrifugal elutriation, lineage subtraction, and fluorescence-activated cell sorting based on high c-kit receptor expression (c-kitBR). We used reverse transcriptase polymerase chain reaction to assay the c-kitBR subset and the subsets expressing low (c-kitDULL) and no (c-kitNEG) c-kit receptor for expression of mRNA encoding hematopoietic growth factor receptors and transcription factors. The c-kitBR cells had approximately 3.5-fold more c-kit mRNA than unfractionated bone marrow cells. The c-kitDULL cells had 47-58% of the c-kit mRNA found in c-kitBR cells and the c-kitNEG cells had 4-9% of the c-kit mRNA present in c-kitBR cells. By comparing mRNA levels in c-kitBR cells (enriched for PHSCs) with those of unfractionated bone marrow, we demonstrated that c-kitBR cells contained low or undetectable levels of mRNA for c-fms, granulocyte colony-stimulating factor receptor, interleukin 5 receptor (IL-5R), and IL-7R. These same cells had moderate levels of mRNA for erythropoietin receptor, IL-3R subunits IL-3R alpha (SUT-1), AIC-2A, and AIC-2B, IL-6R and its partner gp-130, and the transcription factor GATA-1 and high levels of mRNA for transcription factors GATA-2, p45 NF-E2, and c-myb. We conclude from these findings that PHSCs are programmed to interact with stem cell factor, IL-3, and IL-6 but not with granulocyte or macrophage colony-stimulating factor. These findings also indicate that GATA-2, p45 NF-E2, and c-myb activities may be involved in PHSC maintenance or proliferation.

Comparação entre a técnica femoral com dispositivo de hemostasia e a técnica radial em pacientes submetidos à estratégia invasiva precoce

A via de acesso arterial é um importante sítio de complicações após a realização de procedimentos coronários invasivos. Dentre as estratégias para a redução de complicações vasculares, encontra-se estabelecida a eficácia da técnica radial. Os dispositivos de oclusão vascular propiciam maior conforto ao paciente, reduzindo o tempo de hemostasia e repouso no leito. Entretanto, a inconsistência de dados comprovando sua segurança limita sua adoção rotineira como estratégia para redução de complicações vasculares, requerendo evidências de estudos randomizados com metodologia adequada. O objetivo deste estudo foi comparar a incidência de complicações no sítio de punção arterial entre a técnica radial e a técnica femoral com utilização de Angio-Seal em pacientes com síndrome coronariana aguda sem supradesnível do segmento ST submetidos à estratégia invasiva precoce. Trata-se de um ensaio clínico unicêntrico, de não inferioridade, no qual duzentos e quarenta pacientes foram randomizados para a técnica radial ou técnica femoral com utilização de Angio-Seal. O objetivo primário foi a ocorrência de complicações no sítio de punção arterial até 30 dias após o procedimento, incluindo sangramento grave, hematoma >= 5 cm, hematoma retroperitoneal, síndrome compartimental, pseudoaneurisma, fístula arteriovenosa, infecção, isquemia de membro, oclusão arterial, lesão de nervo adjacente ou necessidade de reparo vascular cirúrgico. Em relação às características demográficas e clínicas, houve diferença apenas quanto ao gênero, com presença maior de pacientes do sexo feminino no grupo radial (33,3% versus 20,0%, p=0,020). Não se observaram diferenças entre os grupos quanto ao diagnóstico de admissão, alterações isquêmicas presentes no eletrocardiograma, elevação de marcadores de necrose miocárdica ou escores de risco, bem como quanto à farmacoterapia antitrombótica adjunta e características da intervenção coronária percutânea. A hemostasia foi obtida na totalidade dos procedimentos do grupo radial com a utilização da pulseira compressora seletiva TR Band e em 95% dos procedimentos realizados pela técnica femoral com o Angio-Seal (p=0,029). Exceto pela maior incidência de oclusão arterial no grupo radial comparado ao femoral, não houve diferenças entre os demais desfechos analisados. Segundo o teste de não inferioridade para complicações na via de acesso arterial aos 30 dias, verificou-se que a utilização do Angio-Seal não produziu resultados inferiores ao acesso radial, considerando-se a margem de 15% (12,5% versus 13,3%, diferença -0,83%, IC 95% -9,31 - 7,65, p para não inferioridade <0,001). Os resultados principais deste estudo demonstram que, em uma população de pacientes com diagnóstico de síndrome coronariana aguda sem supradesnível do segmento ST, submetida à estratificação de risco invasiva, a utilização do dispositivo de oclusão vascular Angio-Seal confere ao procedimento efetivado pelo acesso femoral inferioridade na incidência de complicações no sítio de punção arterial aos 30 dias quando comparado ao acesso radial.

Estudo do comportamento dinâmico e do fenômeno de deslocamento em lavadoras de roupas de eixo vertical

Este trabalho apresenta e compara alguns meios para o estudo do comportamento dinâmico de uma lavadora de roupas de eixo vertical durante o processo de extração de água das roupas, também conhecido como centrifugação. Os três métodos utilizados para estudar o comportamento dinâmico de uma lavadora de roupas de eixo vertical foram: analítico, experimental e simulação através de multicorpos. O interesse nesta etapa é devido ao fato dos problemas de vibração de uma lavadora ocorrerem principalmente durante a centrifugação. A modelagem de uma lavadora de roupas durante a etapa de centrifugação permite a obtenção de seu comportamento dinâmico bem como de suas principais características. Um outro tópico estudado neste trabalho foi o fenômeno de deslocamento da lavadora, um dos principais motivos de reclamação dos usuários de lavadoras de roupa automáticas.

Aplicação e avaliação de desempenho de um sistema de otimização em tempo real em uma unidade de produção de propeno.

Com o objetivo de aumentar o lucro de plantas químicas, a Otimização em Tempo Real (RTO) é uma ferramenta que busca determinar as condições ótimas operacionais do processo em estado estacionário, respeitando as restrições operacionais estabelecidas. Neste trabalho foi realizada a implementação prática de um ciclo RTO em um processo de destilação por recompressão de vapor (VRD), propileno-propano, da Refinaria de Paulínia (Petrobras S.A.), a partir de dados históricos da planta. Foram consideradas as principais etapas de um ciclo clássico de RTO: identificação de estado estacionário, reconciliação de dados, estimação de parâmetros e otimização econômica. Essa unidade foi modelada, simulada e otimizada em EMSO (Environment for Modeling, Simulation and Optimization), um simulador de processos orientado a equações desenvolvido no Brasil. Foram analisados e comparados dois métodos de identificação de estado estacionário, um baseado no teste estatístico F e outro baseado em wavelets. Ambos os métodos tiveram resultados semelhantes e mostraram-se capazes de identificar os estados estacionários de forma satisfatória, embora seja necessário o ajuste de parâmetros na sua implementação. Foram identificados alguns pontos estacionários para serem submetidos ao ciclo RTO e foi possível verificar a importância de partir de um estado estacionário para a continuidade do ciclo, já que essa é uma premissa do método. A partir dos pontos analisados, os resultados deste estudo mostram que o RTO é capaz de aumentar o ganho econômico entre 2,5-24%, dependendo das condições iniciais consideradas, o que pode representar ganhos de até 18 milhões de dólares por ano. Além disso, para essa unidade, verificou-se que o compressor é um equipamento limitante no aumento de ganho econômico do processo.

Simultaneous synthesis of work exchange networks with heat integration

The optimal integration of work and its interaction with heat can represent large energy savings in industrial plants. This paper introduces a new optimization model for the simultaneous synthesis of work exchange networks (WENs), with heat integration for the optimal pressure recovery of process gaseous streams. The proposed approach for the WEN synthesis is analogous to the well-known problem of synthesis of heat exchanger networks (HENs). Thus, there is work exchange between high-pressure (HP) and low-pressure (LP) streams, achieved by pressure manipulation equipment running on common axes. The model allows the use of several units of single-shaft-turbine-compressor (SSTC), as well as stand-alone compressors, turbines and valves. Helper motors and generators are used to respond to any demand and excess of energy. Moreover, between the WEN stages the streams are sent to the HEN to promote thermal recovery, aiming to enhance the work integration. A multi-stage superstructure is proposed to represent the process. The WEN superstructure is optimized in a mixed-integer nonlinear programming (MINLP) formulation and solved with the GAMS software, with the goal of minimizing the total annualized cost. Three examples are conducted to verify the accuracy of the proposed method. In all case studies, the heat integration between WEN stages is essential to improve the pressure recovery, and to reduce the total costs involved in the process.

MINLP Optimization Algorithm for the Synthesis of Heat and Work Exchange Networks

This paper introduces a new optimization model for the simultaneous synthesis of heat and work exchange networks. The work integration is performed in the work exchange network (WEN), while the heat integration is carried out in the heat exchanger network (HEN). In the WEN synthesis, streams at high-pressure (HP) and low-pressure (LP) are subjected to pressure manipulation stages, via turbines and compressors running on common shafts and stand-alone equipment. The model allows the use of several units of single-shaft-turbine-compressor (SSTC), as well as helper motors and generators to respond to any shortage and/or excess of energy, respectively, in the SSTC axes. The heat integration of the streams occurs in the HEN between each WEN stage. Thus, as the inlet and outlet streams temperatures in the HEN are dependent of the WEN design, they must be considered as optimization variables. The proposed multi-stage superstructure is formulated in mixed-integer nonlinear programming (MINLP), in order to minimize the total annualized cost composed by capital and operational expenses. A case study is conducted to verify the accuracy of the proposed approach. The results indicate that the heat integration between the WEN stages is essential to enhance the work integration, and to reduce the total cost of process due the need of a smaller amount of hot and cold utilities.

Phosphorylated α-synuclein-immunoreactive retinal neuronal elements in Parkinson's disease subjects

Visual symptoms are relatively common in Parkinson's disease (PD) and optical coherence tomography has indicated possible retinal thinning. Accumulation of aggregated α-synuclein is thought to be a central pathogenic event in the PD brain but there have not as yet been reports of retinal synucleinopathy. Retinal wholemounts were prepared from subjects with a primary clinicopathological diagnosis of PD (N = 9), dementia with Lewy bodies (DLB; N = 3), Alzheimer's disease (N = 3), progressive supranuclear palsy (N = 2) as well as elderly normal control subjects (N = 4). These were immunohistochemically stained with an antibody against α-synuclein phosphorylated at serine 129, which is a specific molecular marker of synucleinopathy. Phosphorylated α-synuclein-immunoreactive (p-syn IR) nerve fibers were present in 7/9 PD subjects and in 1/3 DLB subjects; these were sparsely distributed and superficially located near or at the inner retinal surface. The fibers were either long and straight or branching, often with multiple en-passant varicosities along their length. The straight fibers most often had an orientation that was radial with respect to the optic disk. Together, these features are suggestive of either retinopetal/centrifugal fibers or of ganglion cell axons. In one PD subject there were sparse p-syn IR neuronal cell bodies with dendritic morphology suggestive of G19 retinal ganglion cells or intrinsically photosensitive ganglion cells. There were no stained nerve fibers or other specific staining in any of the non-PD or non-DLB subjects. It is possible that at least some of the observed visual function impairments in PD subjects might be due to α-synucleinopathy.

Retrofit of heat exchanger networks with pressure recovery of process streams at sub-ambient conditions

This paper presents a new mathematical programming model for the retrofit of heat exchanger networks (HENs), wherein the pressure recovery of process streams is conducted to enhance heat integration. Particularly applied to cryogenic processes, HENs retrofit with combined heat and work integration is mainly aimed at reducing the use of expensive cold services. The proposed multi-stage superstructure allows the increment of the existing heat transfer area, as well as the use of new equipment for both heat exchange and pressure manipulation. The pressure recovery of streams is carried out simultaneously with the HEN design, such that the process conditions (streams pressure and temperature) are variables of optimization. The mathematical model is formulated using generalized disjunctive programming (GDP) and is optimized via mixed-integer nonlinear programming (MINLP), through the minimization of the retrofit total annualized cost, considering the turbine and compressor coupling with a helper motor. Three case studies are performed to assess the accuracy of the developed approach, including a real industrial example related to liquefied natural gas (LNG) production. The results show that the pressure recovery of streams is efficient for energy savings and, consequently, for decreasing the HEN retrofit total cost especially in sub-ambient processes.